Marker protein expression cassette capable of inducing regulation and recombinant vector constructed by same and application

A technology for labeling proteins and recombinant vectors, applied in the field of molecular biology, can solve the problems of low transformation efficiency of Bacillus, inability to overcome recombination efficiency, and inability to obtain, etc., to achieve the effect of increasing the quantity, reducing the difficulty of operation, and improving the success rate

Inactive Publication Date: 2017-03-15
NANJING FOODSAFETY BIOTECH CO LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the most widely used method is double crossover homologous recombination. In a simple double crossover homologous recombination system, only the upstream and downstream homology arms and target gene fragments are needed to trans...

Method used

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  • Marker protein expression cassette capable of inducing regulation and recombinant vector constructed by same and application
  • Marker protein expression cassette capable of inducing regulation and recombinant vector constructed by same and application
  • Marker protein expression cassette capable of inducing regulation and recombinant vector constructed by same and application

Examples

Experimental program
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Effect test

Embodiment 1

[0045] In this embodiment, the grac promoter (lactose operon contained) and the toxin protein ccdB recombinant plasmid containing optimized codons were selected. Using the genome of BS001 as a template, through

[0046] Primer 49: acacattaactagacagatctTTTGCCGAAGCTGATCCG (SEQ ID NO.57)

[0047] and primer 50: atggcatgcatcgatagatctGAAGTGTTACCTGTTGACGCGC (SEQ ID NO. 58)

[0048] Amplification of the homology arm upstream of thrC (SEQ ID NO.69)

[0049] Pass Primer 51: acacattaactagacagatctCGAAGCCGACATGCTGTTCT (SEQ ID NO. 59)

[0050] and primer 52: atggcatgcatcgatagatctTTTTCTAAATAAAAACTCCTTTATCTCCA (SEQ ID NO.60) to amplify the homology arm (SEQ ID NO.70) downstream of thrC (PCR conditions: high-fidelity pfu enzyme 1 μL, 10×PCRbuffer 2.5 μL, dNTP (10 mmol / L) 2 μL, Primer (10 μmol / l) 2 μL each, template (Bacillus subtilis BS001 DNA) 1 μL, double distilled water 14.5 μL. Gently blow and mix with a pipette tip. Pre-denaturation at 95°C for 3 minutes; denaturation at 95°C for 40 s...

Embodiment 2

[0052]In this embodiment, the grac promoter (lactose operon contained) and the toxin protein ccdB recombinant plasmid containing optimized codons were selected. Using the genome of BS001 as a template, the homology arm upstream of thrC was amplified by primer 49 and primer 50, and the homology arm downstream of thrC was amplified by primer 51 and primer 52 (the amplification process was the same as in Example 1). pKBS was digested with restriction endonuclease BglⅡ and connected with the upstream homology arm of thrC. Ligated with the downstream homology arm of thrC by restriction endonuclease BamHI digestion. Using pet30A as a template, through

[0053] Primer 53: tgcaaaagccgcagcagatctATGAGCCATATTCAACGGGAAA (SEQ ID NO. 61) and

[0054] Primer 54: atggcatgcatcgatagatctTTAGAAAAACTCATCGAGCATCAAA (SEQ ID NO.62) amplifies the kanamycin resistance gene kan (PCR conditions: high-fidelity pfu enzyme 1 μL, 10×PCR buffer 2.5 μL, dNTP (10 mmol / L) 2 μL, primer (10 μmol / L l) Each 2 μL,...

Embodiment 3

[0056] In this embodiment, the xylose-induced xylA promoter and the toxin protein mazF recombinant plasmid containing optimized codons were selected. Using the genome of BS001 as a template, the homology arm upstream of thrC was amplified by primer 49 and primer 50, and the homology arm downstream of thrC was amplified by primer 51 and primer 52 (the amplification process was the same as in Example 1). Pkbs was digested with restriction endonuclease BglⅡ and connected with the upstream homology arm of thrC. Ligated with the downstream homology arm of thrC by restriction endonuclease BamHI digestion. The vector was then transformed into Bacillus subtilis BS001, and a single colony was obtained on an erythromycin (5 μg / ml) resistant plate, picked and inoculated with LB medium containing erythromycin (5 μg / ml), and treated at 42°C for 12 hours, Erythromycin (5 μg / ml) resistant plates containing X-gal were coated. Pick a blue single colony, inoculate LB medium and culture at 37°...

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Abstract

The invention relates to a marker protein expression cassette capable of inducing regulation and a recombinant vector constructed by the same and an application. A selection marker protein expression cassette capable of inducing regulation is constructed by use of selection marker protein and a regulation induction element; a recombinant vector taking escherichia coli toxic protein as a selection marker is prepared through the selection marker protein expression cassette capable of inducing regulation; the recombinant vector controls to express escherichia coli toxic protein in secondary homologous recombination; the strain only experiencing once homologous recombination is killed or the growth is inhibited while the growth of the strain experiencing twice homologous recombination is not limited, so that the selection efficiency of the strain of twice homologous recombination is improved, the operation flow of gene knock-in or knockout of a bacillus homologous recombination method is simplified, the operation difficulty is lowered, the labor intensity is reduced, and the marker protein expression cassette can be perfectly used for knocking out the bacillus gene and transferring into the bacillus together with exogenous gene.

Description

technical field [0001] The present invention belongs to the field of molecular biology, and actually knocks out and knocks in genes in Bacillus species. Specifically, the present invention relates to a method for knocking out and knocking in Bacillus genes using Escherichia coli toxic proteins ccdB and mazF as screening marker proteins. Background technique [0002] Bacillus (Bacillus) is a class of aerobic, Gram-positive bacteria with endogenous stress-resistant spores. Many of the Bacillus genus can be used as hosts for expressing foreign proteins, such as Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus licheniformis, Bacillus pumilus, giant Bacillus megaterium, Bacillus stearothermophilus, Bacillus alkalophilus, Bacillus coagulans, Bacillus lentus and the like. The above-mentioned Bacillus can be used for industrial production of alkaline protease, amylase, glucanase, xylanase, cellulase, growth hormone, interleukin, penicillin acylase, riboflavin, purine nucleo...

Claims

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Application Information

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IPC IPC(8): C12N15/65C12N15/75
CPCC12N15/65C12N15/75C12N2800/101C12N2840/002
Inventor 陆兆新孟凡强李金良李春燕
Owner NANJING FOODSAFETY BIOTECH CO LTD
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